Flow cytometry Anti-bodies Human - CD3

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

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Found 3 matching solutions for this experiment

PerCP Mouse Anti-Human CD3

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
To perform flow cytometry analysis and to sort BFU-E and CFU-E cells from primary human samples including BM, cord blood, and peripheral blood, we isolated CD45+ cells from mononuclear cells (MNC) derived by Ficoll density gradient separation, followed by positive selection using CD45 magnetic beads.	Ab or appropriate isotype controls (all from BD Biosciences) incubation for 30 minutes in the dark. Cells were washed twice with 40 mL phosphate-buffered saline/0.5% bovine serum albumin, resuspended in 5 mL phosphate-buffered saline/0.5% bovine serum albumin, and stained with the viability marker 7-AAD on ice for 10 minutes in the dark.	The analysis was performed using BD FACSDiva. Sorting was performed using a MoFlo high-speed cell sorter (Beckman Coulter). The number of BFU-E or CFU-E cells in one million MNCs was quantitated.

Upstream tips
To perform flow cytometry analysis and to sort BFU-E and CFU-E cells from primary human samples including BM, cord blood, and peripheral blood, we isolated CD45+ cells from mononuclear cells (MNC) derived by Ficoll density gradient separation, followed by positive selection using CD45 magnetic beads.
Protocol tips
Ab or appropriate isotype controls (all from BD Biosciences) incubation for 30 minutes in the dark. Cells were washed twice with 40 mL phosphate-buffered saline/0.5% bovine serum albumin, resuspended in 5 mL phosphate-buffered saline/0.5% bovine serum albumin, and stained with the viability marker 7-AAD on ice for 10 minutes in the dark.
Downstream tips
The analysis was performed using BD FACSDiva. Sorting was performed using a MoFlo high-speed cell sorter (Beckman Coulter). The number of BFU-E or CFU-E cells in one million MNCs was quantitated.

Manufacturer protocol Publication protocol 1 Relevant paper

APC-Cy™7 Mouse Anti-Human CD3

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
FACS staining was done in FACS buffer (PBS, 1% BSA). For measuring intracellular cytokines, PBMCs were pre-treated for 15 min with the hydroxamic acid derivative GM6001 (100 μM, CALBIOCHEM) to inhibit the shedding of IL-6Rα (4) and stimulated for 4 hours with or without phorbol myristate acetate (50ng/ml, PMA) and ionomycin (1 μg/ml) (both from Sigma Aldrich) in the presence of Golgiplug (BD Bioscience)	For intracellular staining, stained cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Bioscience).	Cells were analyzed on an LSRII® flow cytometer. In most cases, greater than 100,000 events were collected for analysis. Collected data were analyzed using FlowJo software (Tree Star).

Upstream tips
FACS staining was done in FACS buffer (PBS, 1% BSA). For measuring intracellular cytokines, PBMCs were pre-treated for 15 min with the hydroxamic acid derivative GM6001 (100 μM, CALBIOCHEM) to inhibit the shedding of IL-6Rα (4) and stimulated for 4 hours with or without phorbol myristate acetate (50ng/ml, PMA) and ionomycin (1 μg/ml) (both from Sigma Aldrich) in the presence of Golgiplug (BD Bioscience)
Protocol tips
For intracellular staining, stained cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Bioscience).
Downstream tips
Cells were analyzed on an LSRII® flow cytometer. In most cases, greater than 100,000 events were collected for analysis. Collected data were analyzed using FlowJo software (Tree Star).

Manufacturer protocol Publication protocol 1 Relevant paper

PE anti-human CD3 Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
	Ab incubation for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature. After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software.

Protocol tips
Ab incubation for 20 min at 37 °C. Annexin V was then added and stained for 15 min at room temperature. After incubation, BMMCs were washed, centrifuged, measured by a FACSCanto II (BD Biosciences) and resulting data were analysed using FlowJo software.

Manufacturer protocol Publication protocol 1 Relevant paper

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